Background We are testing several approaches, including gene therapy, small molecule screening for drug discovery, and retinal reconstruction. All strategies are guided by the knowledge of molecular mechanisms of photoreceptor development, homeostasis, and disease acquired through other projects (see EY000450-473-475) Results 1. Gene therapy In collaboration with Drs. P. Colosi and T. Li, we have initiated pre-clinical studies for the treatment of Leber congenital amaurosis (LCA) caused by CEP290 mutations and of retinitis pigmentosa (RP) caused by mutations in the RPGR and RP2 genes. N-NRL has focused on animal models and genetic characterizations, whereas Peter Colosi's unit is involved in AAV-based gene therapy (see Colosi report). As CEP290 is a large gene, we are working with Colosi and Li groups to test different AAV constructs and lenti-viruses. As a novel tool for gene therapy, we are testing AAV vectors containing 0.3 kb of Nrl promoter/enhancer that can specifically direct the target gene to both developing and mature rod photoreceptors. In collaboration with Drs. Sam Jacobson, Gus Aguirre and Bill Hauswirth, we have proved the efficacy of gene augmentation therapy in two blinding canine photoreceptor disease models for the common X-linked form of retinitis pigmentosa, caused by mutations in the RPGR gene (1). Our results provide a path for translation to human treatment. 2. Drug discovery We are developing reagents and protocols for high throughput screens of small molecules, to discover new drugs to prevent photoreceptor degeneration. We are using zebrafish as animal model, developing in vitro biomaterial-based cell cultures, and plan to use iPS cells for small molecule testing. Candidate molecules will be further investigated in preclinical studies using our well-characterized mouse models of retinal degeneration. Our small molecule screens will search for chemicals that can either influence photoreceptor differentiation o rescue photoreceptor degeneration. We are using three zebrafish transgenic lines. In TalphaCP-GFP and Rho-GFP lines, GFP expression is restricted to cones or rods, respectively. In a pilot screen of the NCI Diversity II library of about 1800 compounds using the TalphaCP-GFP reporter, we identified seven candidate compounds that increase the reporter fluorescence and two compounds that cause a decrease in fluorescence intensity, seemingly promoting and preventing photoreceptor differentiation, respectively. The Pde6cw59 line contains a mutation in the cone phosphodiesterase C. As a result, homozygous embryos exhibit rapid degeneration of cone photoreceptors by 4 dpf. Homozygous mutant embryos, which are positive for the fluorescent marker, will be subjected to the NCI Diversity II library. Candidate compounds will be evaluated further to determine their dose curve, IC50 and mechanism of action. Other zebrafish lines carrying mutations in genes that are associated with retinal degeneration are being generated or acquired from collaborators. We have established that Collagen Vitrigel (from Toshiaki Takezawa) is a suitable biomaterial substrate for culturing developing rods and favors cell survival, establishment of neuronal morphology, and expression of rod markers, including rhodopsin. We are developing a biomaterial-based co-culture model that includes iPSC- and ESC-derived photoreceptors with RPE-derived soluble molecules or cells. The use of biomaterials to create an environment that more closely mimics in vivo photoreceptor-RPE architecture is a significant improvement over current two-dimensional or solution-based culture systems and sets the stage for their use in large scale screening assays and as transplantable scaffolds for retinal cell replacement therapies. 3. Use of iPSCs and ESCs to develop therapies We previously showed that Nrl-expressing newborn rod photoreceptors can differentiate and integrate in degenerating mouse retina, thus suggesting the feasibility of retinal repair by photoreceptor replacement. Since then, others and we have been pursuing avenues to increase the efficiency of integration and to generate photoreceptors from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). More recently, we tested the potential of human ESCs to produce retinal neurons in vivo (2). Furthermore, we have successfully developed protocols to grow human iPSCs and ESCs and differentiated these into retinal neurons. We have generated several fluorescence reporter constructs that will now be stably integrated into stem cells using PiggyBac or zinc-finger technology. Lineage-restricted cells (as determined by the expression of specific promoter construct) will be used for transplantation in the degenerating retina. Calcium imaging and patch clamp recording are being used to functionally characterize the stem cell-derived photoreceptors in vitro and after transplantation in the recipient mouse. Based on RNA-Seq analysis of lineage-restricted cells, we will identify cell surface markers that will allow us to purify sub-populations of differentiating cells for transplantation and to establish which one has the best therapeutic effect.